The present invention relates in general to the cryopreservation of cells and more specifically to the large scale cryopreservation of porcine hepatocytes.
The liver is an essential organ for the survival of humans because it serves a variety of important functions related to macromolecular synthesis, energy generation and storage, catabolism and disposal of toxic substances and waste products of intermediary metabolism. The biochemical functions in which the liver plays a major role include the metabolism of amino acids, ammonia, proteins, carbohydrates and lipids; biochemical oxidation; and metabolism and detoxification of drugs, vitamins and hormones. Given the aforementioned, acute or chronic liver impairment, carries a poor prognosis.
Treatment for end stage acute or chronic hepatic failure may be divided into two categories, namely the use of artificial liver support systems (ALS) and orthotopic liver transplantation (OLT). ALS methods include hemoperfusion, hemodialysis, hemofiltration and plasmapheresis. Unfortunately, these methods do not improve the survival because they do not provide metabolic support.
In contrast, transplantation has a 50-90% 1 year survival rate. Unfortunately however, donor livers are in high demand but in short supply. 15-30% of patients have been reported to die before a matching donor is found.
Lastly, ALS and hepatocyte transplantation methods have been studied. Specifically, isolated hepatocytes have been investigated for use in artificial liver devices to compensate for the loss of metabolic function in the damaged liver. Hepatocyte transplantation as opposed to full liver transplantation has also been investigated as an alternative treatment modality. Both techniques to be effective however, require large scale cryopreservation of viable hepatocytes.
Shinichi Kasai and Michio Mito in "Large-Scale Cryopreservation of Isolated Dog Hepatocytes", Cryobiology 30, 1-11, 1993, describes an isolation method wherein the viability of preserved hepatocytes is 75+/-3% and the estimated recovery rate is approximately 50%. Moreover, preserved hepatocytes showed 20-50% of the metabolic activity of fresh cells as assessed by ammonia and fructose loading tests, ATP content, C-14 leucine uptake. While this technique is a beginning, it leaves much room for improvement in both yield and viability of hepatocytes.
Thus it is a primary objective of the present invention to provide for a treatment method for acute and chronic hepatic failure which provides for a high yield of viable cryopreserved hepatocytes. A further objective is to provide for a high yield of viable cryopreserved hepatocytes to allow for quality control and bioburden testing prior to use.